Isolation and molecular identification of extracellular lipase-producing bacillus species from soil | Abstract
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Isolation and molecular identification of extracellular lipase-producing bacillus species from soil

Author(s): Khataminezhad Mohammad Reza, Nuhi Ashrafalsadat, Razavi Mohammad Reza, Nejadsattari Taher and Nazemi Ali

Microbial lipases are the most valuable Biocatalysts with Industrial applications that show a large variety of enzymatic properties among microbial populations. This study aimed to isolate extracellular lipase-producing Bacilli and their molecular identification in the soils of western Mazandaran, Iran. A total of 50 soil samples with bacterial lipolytic potential were collected from the depth of 10-15 cm of soils in the rural areas of western Mazandaran, Iran. The samples were directly cultured on tributyrin agar medium with Lipidic glycerol butyrate substrate after heat treatment and preparation of serial dilutions. DNA was isolated from bacillus isolates with a clear zone. The molecular identification of isolates was carried out by using 16S rDNA Real Time PCR, High Resolution Melting (HRM), direct sequencing, and sequence alignment in BLAST. The primary properties of lipase enzymes produced by isolates were determined using supernatant of medium culture by spectrophotometer measurement of enzymatic activity at different temperatures, different concentrations of substrate, and relative molecular weight determination of enzymes by SDS-PAGE. Total of 5 gram-positive bacilli was isolated. After identification of biochemical and morphological characteristics, sequence alignment of their 16S rDNAs, the strains were identified as B. megaterium 37-1, Bacillus safensis 1-1, Bacillus pumilus KN-Lip2, Bacillus subtilis KN-Lip3, and Lysinibacillus fusiformis KN-Lip4 in Genbank database. The highest lipase activity of each strain was determined as 626.1069 u/ml and 422.5517 u/ml for B. megaterium 37-1 and B. safensis 1-1, respectively. The relative molecular weight of lipases produced by both strains was about 55 KDa. Vmax and km parameters were calculated by Michaeilis-Menthen hyperbola and Lineweaver-Burk plots. The Vmax value of lipase enzyme produced by B. megaterium 37-1 at 37°C was higher than that produced by B. safensis 1-1. The km values of B. megaterium 37-1 were higher and lower than that of B. safensis 1-1 at 37°C and 55°C, respectively. In this study, lipolytic bacillus isolates were collected and identified from soil of various environments in the northern part of Iran. Among 5 new strains found in this study, B. megaterium 37-1 showed a higher enzymatic activity in compare to other strains.